FTIR microspectroscopy study of compositional changes in biological samples



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Fourier Transform Infrared (FTIR) microspectroscopy has emerged as a powerful technique for analyzing biological samples. Obtaining spatially resolved molecular information from the samples is one of the major advantages of this technique. Qualitative, as well as quantitative analysis of the sample, is possible with the use of this technique. It gives information regarding the intrinsic molecular chemistry of the sample. It could be applied for the analysis of wide range of samples. In this study, we used FTIR microspectroscopy to investigate the changes in the chemical composition of mouse adipose tissue upon oxygen plasma treatment and cellulose substrate upon plasma treatment and grafting of vinyl laurate. The first chapter describes the instrumentation and principle of operation of FTIR microspectroscopy and microwave plasma. Moreover, it provides a description of adipose tissue and cellulose. The chapter also covers reactive oxygen species (ROS), their functions and their role in oxidative stress. In the second chapter, the effect of ROS on lipids and proteins of mouse white adipose tissue was studied using FTIR microspectroscopy. Microwave plasma was used to induce oxidative damage to the biomolecules of white adipose tissue (WAT) of mouse through insitu production of ROS. The analysis of the IR spectra extracted from the infrared (IR) images of adipocyte and ECM of the mouse white adipose tissue showed a significant effect of ROS on lipids. Mainly, unsaturated lipids were found to be highly affected by ROS as a drastic decrease in the area of the band attributed to the olefinic (=CH-) vibration from lipid was observed. Similarly, changes associated with saturated lipids were also observed. Along with the decrease in the area of bands assigned to lipids, significant increase in the area of carbonyl (C=O) band was observed. However, amide bands from proteins did not change significantly, indicating that proteins are comparatively more resistant to ROS than lipids. Chemimaps and band ratio images were developed from the FTIR image recorded after each treatment. Chemimaps clearly showed decreasing concentrations of olefinic group and increasing concentration of the carbonyl group. Similarly, band ratio images showed decreasing intensity of olefinic/lipid ratio and increasing intensity of carbonyl/lipid ratio. In the third chapter, grafting of vinyl laurate monomer on the regenerated cellulose film and cotton fibers was studied using FTIR microspectroscopy. The grafting of monomer on the cellulose films and cotton fibers was initiated by means of microwave plasma treatment. The analysis of spectra extracted from the FTIR images of the monomer grafted samples showed the presence of additional peaks at 1735, 2925 and 2855 cm-1. Comparison of correlation coefficient between the spectra of the control and those of treated samples showed significantly higher correlation in treated samples compared to control samples. Chemical distribution maps showed non-uniform distribution of vinyl laurate on the cellulose film as indicated by a non-uniform distribution of carbonyl group on the FTIR image of the cellulose film. In the final chapter, a brief summary of three chapters is included. It also describes the importance of FTIR imaging and future directions of the projects described in chapters 2 and 3. Overall, the results showed that FTIR imaging is effective in analyzing animal tissue as well as plant materials. This shows a wide range of application of FTIR imaging and could be used as a rapid and sensitive technique to monitor subtle changes in biomolecules. The simplicity of the sample preparation, non-destructiveness, and ease in obtaining results has made it an excellent tool for the analysis of biological samples. Overall, the present study is further important as it sheds light on the compositional changes occurring in the macromolecular content of biological samples, which could be useful for further studies.



FTIR Microspectroscopy, Microwave Plasma, White Adipose Tissue, Adipocyte, Extracellular Matrix, Chemimaps, Cellulose, Vinyl Laurate, Cotton Fibers, Cellulose Films, Reactive Oxygen Species, Oxidative Stress